Produção embrionária in vitro a partir de oócitos bovinos selecionados por corantes vitais e maturados em meio suplementado

Abstract

Culture conditions in vitro and oocyte competence are the major factors related to embryonic in vitro production low results. Therefore, this study aimed to evaluate in vitro embryo production rates of bovine oocytes selected with Brilliant Cresyl Blue (BCB) or Trypan Blue (TB), matured in supplemented medium with insulin and / or IGF I. Ovaries were obtained from abattoir and the oocytes were collected by follicular aspiration using syringe and needles. Grade I and II oocytes were selected under stereomicroscope and stained with BCB (SIGMA®) for 60 minutes at 26 μM or for 10 minutes using TB at 0.16% (VETEC®), at 38,5 °C, 5% CO2, 90% humidity. After staining, oocytes were washed 3 times with DMPBS (NUTRICELL®) and separated in stained (BCB+ or TB+) or unstained (BCB- or TB-). Only BCB+, TB+ and TB- oocytes underwent for subsequent steps of in vitro maturation in medium without supplementation (control group), supplemented with IGF I (10 ng/ mL), supplemented with insulin (12 IU/ mL) and supplemented with IGF I (10 ng / mL) and insulin (12 IU/ mL), in vitro fertilization and in vitro culture. At D8, oocytes were evaluated for cleavage, yield and quality of blastocysts, according to the stain and maturation medium. As for BCB+ oocytes, we observed that there was no statistical difference in production and quality rates of blastocysts with supplemented maturation medium. TB+ and TB- oocytes showed good embryo development, around 71% (TB+) e 89% (TB-) indicating that TB is not a very efficient tool for oocyte selection. Regarding maturation medium used for oocytes undergoing TB, it was noted that supplementation with insulin provided better results as the production rate and quality of these blastocysts. We can conclude that BCB is an effective dye for the selection of the most competent oocytes, and that insulin or IGF I supplemented maturation medium improves quality and production rate of blastocysts, but in oocytes that had already been selected for development ability, this increase is not significant.